Cardiovascular disease (CVD) is the leading cause of death among the elderly in the United States. Since the percentage of Americans over the age of 75 years is expected to increase from 6% in 2000 to 11 % in 2050, the number of persons dying from CVD will likely rise dramatically in the coming years. In previous work, this team of investigators demonstrated that acute and chronic particulate air pollution exposures are associated with specific cardiovascular outcomes, such as decreased heart rate variability, arterial stiffness, and ST-segment depression, resulting in increased death rates for CVD. DNA-methylation changes have been found to be involved in inflammation, endothelial function, and atherosclerosis. Initial observations in experimental models and humans have shown that air particles and metals, which are constituents of particulate pollution, alter DNA methylation status. Whether the increased risk of CVD observed in subjects exposed to air particles and toxic metals (such as arsenic, cadmium and lead) is mediated by alterations of DNA-methylation patterns is unresolved. The goal of this proposal is to determine whether exposure to air particles and toxic metals (arsenic, cadmium and lead) increases CVD risk by altering global and/or gene specific methylation in elderly subjects over time. Specifically, this proposal will evaluate: 1) the association of the exposure with global DNA methylation in Alu and LINE-1 sequences, and with hypermethylation or hypomethylation in specific genes (ICAM-1, IFN-gamma, p16) involved endothelial function and atherosclerosis;and 2) the association between the above global and gene specific DNA-methylation changes and CVD-related outcomes (Heart-rate variability, Pulse Doppler measurements of atherosclerosis, ST segment elevation/depression, biological markers of inflammation/endothelial function). As a secondary aim, this initiative will explore whether: 1) global or gene-specific methylation status modifies the association of air particles and toxic metals (Pb, As, Cd) with CVD-related endpoints;and 2) the association between exposure and methylation status is modified by plasma levels or intakes of dietary factors (folate, Vitamin B6 &B12), plasma homocysteine levels, and SNPs in DNA methylation genes (MTHFR, cSHMT). The study design, based on an existing cohort of 700 elderly subjects in Eastern Massachusetts, will allow for baseline and longitudinal analysis of air particles, metal biomarkers, DNA methylation patterns and CVD phenotypes, therefore all hypotheses will be tested cross-sectionally and longitudinally. Quantitative DNA methylation analysis, based on PCR amplification and pyrosequencing of bisulphite-treated DNA, will be performed for each study subject on three blood DNA samples collected over a long time span.